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1 CH50 Eq Enzyme Immunoassay Kit For in-vitro diagnostic use only Product code: MK095 The Binding Site Group Ltd, PO Box 11712, Birmingham B14 4ZB, UK Telephone: +44 (0) Fax: +44 (0) FDA (USA) Information Analyte Name Complement components immunological test system Complexity Cat. High (goat) antibodies to TCC CH50 Eq Activator 10mL, contains human gammaglobulins and murine monoclonal antibodies in phosphate buffered saline (PBS) with 0.035% ProClin 300. Tween-20 is a registered trademark of ICI Americas Inc. ProClin is a registered trademark of Rohm and Haas Company. 4.2 Materials required but not provided Timer (60 minute range) Calculator or other computational method to validate the assay Clean, unused microassay plates and/or test tubes and racks Container for wash buffer dilution Wash bottle or other immunoassay washing system Adjustable multichannel pipette (8 or 12 channels) or repeating micropipettes (optional) Clean pipettes, 1 ml, 5 ml, and 10 ml Micropipettes and pipette tips Plate reader capable of optical density A 450 readings between 0.0 and Deionized or distilled water Incubator or microassay plate heater (37 C) Water bath (37 C) 5 WARNINGS AND PRECAUTIONS 1 INTENDED USE The CH50 Eq EIA measures the total classical complement pathway activity in human serum and allows detection of a deficiency of one or more of the complement components C1 through C9. 2 SUMMARY AND EXPLANATION The binding of C1q component of C1 to immune complexes triggers the classical complement pathway. This activation results in a cascade of enzymatic and nonenzymatic reactions, culminating in the formation of terminal complement complexes (TCC). Under standard conditions the level of TCC that can be generated in a serum is a quantitative expression of the serum s total classical complement activity. The traditional method for measuring the total classical complement activity in serum is the CH50 test 1. This test is a lytic assay, which uses antibody-sensitized sheep erythrocytes (EA) as the activator of the classical complement pathway and various dilutions of the test serum to determine the amount required to give 50% lysis. The percent haemolysis is determined spectrophotometrically. The CH50 test is an indirect measure of TCC, since the TCC themselves are directly responsible for the haemolysis that is measured. The CH50 Eq EIA provides a direct measure of the total classical complement activity in serum by quantifying the amount of TCC generated under standard conditions. The CH50 Eq EIA uses a monoclonal antibody to a unique neoantigen to capture the TCC analyte. Since both the CH50 Eq EIA and the CH50 test rely on the generation of TCC and correlate, the CH50 Eq EIA s results are expressed in CH50 unit equivalents per millilitre. 3 PRINCIPLE The CH50 Eq EIA for quantifying the total classical complement activity in human serum involves three basic procedures: (1) complement activation; (2) sample dilution; and (3) assay for terminal complement complexes (TCC). To activate the classical complement pathway, undiluted human serum samples and the controls are added to test tubes (or uncoated microassay wells) containing the activator. During incubation the classical pathway of complement is triggered and TCC are generated. In the second step, the activated sera are diluted in uncoated microassay wells or test tubes and dispensed, together with kit standards, directly into a microassay plate. The TCC present in the activated samples bind to the monoclonal antibodies coating the surface of the microassay wells. In the third step, the TCC microassay plate is washed and loaded with an HRPconjugate, which will bind to the bound TCC. After washing, the TCC microplate is loaded with a chromogenic enzyme substrate. After incubation a reagent is added to stop colour development. The absorbencies (A 450 values) generated with the controls, kit standards, and test specimens are measured spectrophotometrically. The colour intensity of the reaction mixture is proportional to the concentration of TCC present and to CH50 units. Using the kit standard curve, assay results are expressed in CH50 unit equivalents per millilitre (CH50 U Eq/mL). 4 REAGENTS 4.1 Materials provided CH50 Eq Standard A, B, C, D, E 1 x 1.5mL of each standard (A-E) Each contains human serum with assigned CH50 unit equivalents per ml, (U Eq/mL), protein stabilizers CH50 Eq Normal Control 3 x 100µL, lyophilized. When reconstituted, each contains human serum CH50 Eq Low Control 3 x 100µL, lyophilized. When reconstituted, each contains human serum Anti-TCC Coated Wells 96-well microassay plate with retainer and holder consisting of 12 eight-well strips coated with a mouse monoclonal antibody in a resealable foil pouch CH50 Eq Stop Solution 12mL, contains 1N (4%) hydrochloric acid CH50 Eq Wash Buffer 20x concentrate, 2 x 50mL, contains phosphate 7 SPECIMEN COLLECTION buffered saline (PBS), 1.0% Tween-20, and 0.035% ProClin CH50 Eq Sample Diluent 50mL, Contains phosphate buffered saline (PBS), 0.05% Tween-20, 2.5% protein stabilizers, 0.035% ProClin CH50 Eq TMB Substrate, 12mL, contains 3,3,5,5 tetramethylbenzidine (TMB) and Hydrogen Peroxide (H 2 O 2 ) CH50 Eq Conjugate 7mL, Contains horseradish peroxidase-conjugated Insert Code: E095, Version: 21 st September 2009, Page 1 of For in-vitro diagnostic use. 5.2 Treat specimen samples as potentially biohazardous material. Follow Universal Precautions when handling contents of this kit and any patient samples Wear suitable protective clothing, gloves, and eye/face protection when handling contents of this kit Use the supplied reagents as an integral unit prior to the expiration date indicated on the package label Store assay reagents as indicated Do not use coated strips if pouch is punctured ProClin 300 is used as a preservative. Incidental contact with or ingestion of buffers or reagents containing ProClin can cause irritation to the skin, eyes or mouth. Use good laboratory practices to reduce exposure. Seek medical attention if symptoms are experienced The stop solution is considered corrosive and can cause irritation. Do not ingest. Avoid contact with eyes, skin, and clothing. If contact is made, immediately rinse affected area with water. If ingested, call a physician Each donor unit used in the preparation of the standards and control sera of this product was tested by an FDA-approved method for the presence of antibody to human immunodeficiency virus (HIV1 and HIV2) and to hepatitis C virus, as well as for hepatitis B surface antigen. Since no test method can offer complete assurance that infectious agents are absent, these reagents should be handled at Biosafety Level 2 as recommended for any potentially infectious human serum or blood specimen in the Centers for Disease Control/National Institutes of Health manual Biosafety in Microbiological and Biomedical Laboratories, Use of multichannel pipettes or repeat pipettors is recommended to ensure timely delivery of reagents For accurate measurement of samples, add samples and standards precisely. Pipette carefully using only calibrated equipment Proper collection and storage of test specimens are essential for accurate results (see SPECIMEN COLLECTION and STORAGE) Avoid microbial or cross-contamination of specimens, reagents, or materials. Incorrect results may be obtained if contaminated Test each sample in duplicate Do not use a microassay well for more than one test Using incubation times and temperatures other than those indicated in the ASSAY PROCEDURE section may give erroneous results The TMB substrate must be protected from light during storage and incubation. Avoid contact with eyes, skin, and clothing. If contact is made, immediately rinse affected area with water Do not allow microassay wells to dry once the assay has begun When adding or aspirating liquids from the microassay wells, do not scrape or touch the bottom of the wells Heat-inactivated, hyperlipaemic or contaminated specimens may give erroneous results To avoid aerosol formation during washing, use an apparatus to aspirate the wash fluid into a bottle containing household bleach A wash bottle or automated filling device should be used to wash the plate (ASSAY PROCEDURE, Step 9). For best results, do not use a multichannel pipette to wash the microassay plate Dispose of containers and unused contents in accordance with Federal, State and local regulations. 6 STORAGE Store unopened kit at 2 8 C. After the kit is opened, the 20X wash solution concentrate may be stored at 2 30 C. After selecting the reagents or materials to be used in the assay, return the unused reagents immediately to their appropriate storage temperatures. Bring reagents and materials to room temperature (15 30 C) before use. INDICATIONS OF INSTABILITY OR DETERIORATION OF REAGENTS: Cloudiness or discoloration of the diluted wash solution indicates a deterioration of this reagent. If this occurs, the solution should be discarded. The activator may contain particles. This is normal and does not affect performance of the assay. Use serum as the specimen in this assay. Plasma is not acceptable. Handle and dispose of all specimens using Universal Precautions. The proper collection, processing, storage, and shipment of specimens is essential, since complement may be activated in improperly handled specimens. This could

2 lead to erroneous results. Serum specimens should be collected aseptically and prepared using standard techniques for clinical laboratory testing. Samples can be stored up to 2 hours at room temperature, up to 4 hours on ice, up to 3 days at 4 C, and at -70 C or below for long-term storage. Do not subject samples to more than 6 freeze/thaw cycles. Frozen specimens should be tested as soon as possible after thawing or stored on ice (for no longer than four hours) until assayed. Specimens should be packed in excess dry ice for shipping. Specimens that arrive thawed at the testing facility may be compromised. After receiving a shipment, samples can be stored at -70 C or below. 8 REAGENT PREPARATION After removing the needed reagents and materials, return the unused items to their appropriate storage temperatures (see STORAGE). Bring all reagents and materials for the assay to room temperature (15 30 C) before use Wash solution Mix the 20X wash solution concentrate by inverting the bottle several times. If the 20X wash solution concentrate has been stored at 2 8 C, crystals may have formed. To dissolve the crystals, warm the bottle in a C water bath until all crystals have dissolved and follow by mixing thoroughly. Prepare the wash solution by diluting the entire contents of one of the bottles of 20X wash solution concentrate up to one litre with distilled or deionized water. Mix thoroughly. The wash solution is stable for 30 days when stored in a clean container at 2 8 C. If discoloration or cloudiness occurs, discard the reagent Selecting the microassay strips Determine the number of wells required for the assay. It is recommended that the blank wells, controls, and standards be tested in duplicate. Remove the strip retainer from the assembled plate. Remove the unneeded strips and place them in the storage bag, reseal the bag and return it to storage at 2-8 C. Secure the strips to be used in the assay Specimen dilution Caution: Treat all specimens as if potentially infectious. Do not use heatinactivated, contaminated specimens, or improperly stored specimens. Serum specimens are required for this assay. Serum samples should be thawed quickly by placing them briefly in a 37 C water bath. Immediately upon thawing, place the samples on ice until the activation step (see ASSAY PROCEDURE). Do not dilute the test samples prior to activation Preparation of normal and low controls To one vial each of the lyophilized normal and low control add 100µL of deionized or distilled water. Mix briefly to ensure reconstitution and let sit for fifteen (15) minutes at room temperature. Vortex again and place on ice until ready for the activation step. 9 ASSAY PROCEDURE Read entire product insert before beginning the assay. See REAGENT PREPARATION and WARNINGS AND PRECAUTIONS before proceeding Activation of samples and controls Mix the activator, which contains readily suspendable particles, by swirling the bottle before use. Swirl frequently during this step to ensure suspension of the particles. Add 86µL of activator to the appropriate number of test tubes or microassay wells.* Next add 14µL of undiluted serum specimen, normal control, or low control to individual test tubes or microassay wells containing the activator. Mix carefully. Cover to minimize evaporation. Incubate at 37 C for sixty (60) minutes. (FOR YOUR CONVENIENCE: If you wish to batch samples, after activation you may store the undiluted, activated samples at -20 C or colder for up to 14 days before proceeding to the next steps.) * The number of test tubes or microassay wells required for activation equals the number of test samples plus two (2) additional ones for activation of the two controls Dilution of activated samples and controls After activation, dilute the activated samples and activated controls 1:200 in specimen diluent in clean, unused microassay wells or in test tubes. 9.3 Prepare the microassay strips as follows: a. Rehydrate microassay wells by filling each with wash solution ( µL/well) to each well using a wash bottle or an automated device. b. Incubate at room temperature (15 30 C) for two minutes. c. Remove the liquid from each well. d. Invert the plate and tap firmly on absorbent paper repeatedly to remove any remaining liquid. 9.4 Add 100µL of specimen diluent to the duplicate wells that will be used to blank the plate reader. 9.5 Add 100µL of each ready-to-use standard (A-E) to duplicate wells. Note that the standards have already been diluted and are ready to use. 9.6 Add 100µL of the diluted, activated normal and low controls to duplicate wells. 9.7 Add 100µL of each diluted specimen in duplicate to its assigned microassay wells. 9.8 Incubate at room temperature (15 30 C) for 60 ±1 minutes. 9.9 Wash the microassay wells as follows: Note: A multi-channel pipette is not recommended for this step. a. After the incubation in step 8 (or in step 12 below) remove the liquid from each well. b. Using a wash bottle, automated plate washer, or other washing device, fill each well with the wash solution ( µL/well). c. Incubate the wells for 1 minute at room temperature (15-30 C). d. Remove the liquid from each well. e. Fill each well with wash solution ( µL/well). f. Remove the liquid from each well. g. Repeat steps e-f five additional times. h. After the seventh wash cycle, invert the plate and tap firmly on Insert Code: E095, Version: 21 st September 2009, Page 2 of 14 absorbent paper repeatedly to remove any remaining fluid Using a multichannel or repeating pipette, dispense 50µL of conjugate into each washed test well, including the blank well(s) Incubate the microassay strips at room temperature (15 30 C) for 60 ±1 minutes Wash the microassay wells after the 60-minute incubation, as described in step Immediately following the wash procedure, dispense 100µL of the TMB substrate solution into each well, including the blank(s) Incubate the microassay strips at room temperature (15 30 C) for 15±1 minutes Add 100µL of stop solution to each well to stop the enzymatic reaction. The stop solution should be added to the wells in the same order and at the same rate as was the substrate solution. Gently tap the plate to disperse the colour development evenly Determine the absorbance reading at 450nm (A 450 value) for each test well within one hour after the addition of the stop solution, making the necessary blank correction. No reference filter is required Dispose of the remaining diluted specimens, activator, controls, and the used microassay strips (see WARNINGS AND PRECAUTIONS, step 23) Keep the strip holder and strip retainer for future use. 10 RESULTS AND QUALITY CONTROL 10.1 Quality control Good laboratory practice recommends the use of controls to ensure that the assay is performing properly. Each CH50 Eq EIA kit contains normal and low controls that can be used for this purpose. These controls should be tested at least once for each batch of specimens, i.e., for each separate activation run. The controls, when used as instructed, should give CH50 unit equivalent values within the ranges specified on their vial labels. Since these controls are to be activated, diluted, and tested exactly like a typical specimen, they serve as both controls and reference standards for each activation and CH50 Eq EIA run. External controls, prepared by your laboratory, may also be used to help ensure that the assay is performing properly. In addition, the product insert requires that the standard curve generated with the kit A-E standards meet stringent validation requirements (see INTERPRETATION OF RESULTS). Standards should be tested in duplicate for each assay run. If the assay does not meet these requirements, repeat the assay or contact your local Binding Site representative for technical assistance Interpretation of results Use of the Standard Curve: The standard curve for the CH50 Eq EIA is generated using the blank-subtracted A 450 values for each standard (on the y axis) and the assigned concentration for each standard (along the x axis). The standard curve must meet the validation requirements. Most computers and calculators are capable of performing this calculation. An example of a typical standard curve is shown in Figure 1. Calculation of Actual CH50 U Eq Level in Patient Specimens: Sample values are calculated from the standard curve using linear regression analysis. For test samples that were activated and diluted 1:200 prior to testing, the CH50 U Eq/mL can be read directly from the standard curve. The values (U Eq/mL) for the normal and low controls can also be read directly from the standard curve, since you have also diluted them 1:200. In order to obtain accurate CH50 determinations for test specimens that yield A 450 values greater than that of the CH50 Eq EIA Standard E (or that yield A 450 values less than that obtained with Standard A), the test specimens may be re-assayed at a different dilution so that their new A 450 values will be within these limits. In all repeat assays the standards and controls must also be tested. If a sample is run in the assay at a dilution other than 200-fold, the CH50 level of the sample is determined by correcting for the difference between the dilution examined and the usual 200-fold dilution. For example, if a sample is diluted 100-fold rather than the usual 200-fold, and the linear regression curve yields a concentration of 100 CH50 U Eq/mL, then the actual CH50 U Eq/mL in the sample is 50 (i.e., 100 CH50 U Eq/mL divided by 2). Example of standard curve Figure Validation Calculate the CH50 U Eq/mL for the normal and low controls. Also determine the correlation coefficient (r) and the y-intercept (b) of the derived best fit Iinear curve for the values obtained with the A-E Standards. To validate the assay, the r, b, and control values must be within the ranges shown below: Correlation coefficient (r): > 0.96 y-intercept (b): ( ) to (+) Slope (m) Normal & low control values: Within ranges indicated on their respective vial labels Limitations of the procedure The CH50 Eq EIA has been used to test specimens collected as serum. The clinical

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